High activity growth factor mutants

ABSTRACT

The invention relates to novel biosynthetic growth factor mutants which exhibit improved biological activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/094,227 filed May19, 2008, which is a 35 U.S.C. 371 National Phase Entry Application fromPCT/EP2006/011074, filed Nov. 17, 2006, which claims the benefit ofEuropean Patent Application No. 05025261.8 filed on Nov. 18, 2005, thedisclosures of which are incorporated herein in their entirety byreference.

The invention relates to novel recombinant biosynthetic growth factormutants exhibiting improved biological activity. Said improved proteinactivity is achieved by the substitution of specific amino acids of theoriginal growth factor proteins which are naturally occurring proteinsof the transforming growth factor-beta superfamily of signallingmolecules. The recombinant proteins provided herein are particularlysuitable for inducing regeneration, growth stimulation and-differentiation of various cells, tissues and organs. The inventionalso relates to nucleic acid molecules coding for said recombinantprotein mutants, expression vectors and host cells containing thenucleic acid molecules, antibodies directed against said proteinmutants, pharmaceutical compositions and methods for producing thegrowth factor mutants.

The transforming growth factor-beta (TGF-beta) superfamily of proteinscomprises more than 35 members including TGF-betas, bone morphogeneticproteins (BMPs), activins, inhibins and growth/differentiation factors(GDFs). TGF-beta superfamily proteins promote cell proliferation and-differentiation as well as tissue formation and are relevant for a widerange of medical treatment methods and applications. These dimericmolecules act through specific receptor complexes that are composed oftype I and type II serine/threonine receptor kinases. The receptorkinases subsequently activate Smad proteins, which then propagate thesignals into the nucleus to regulate target gene expression. Smadindependent signalling pathways are also initiated by these receptorsand result in induction of the MAP Kinase pathway. Smads are a uniquefamily of signal transduction molecules that can transmit signalsdirectly from the cell surface receptors to the nucleus, where theyregulate transcription by interacting with DNA binding partners as wellas transcriptional coactivators and corepressors.

The members of this protein family are initially synthesized as largeprecursor proteins which subsequently undergo proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus, thus releasing the C-terminal mature protein part from theN-terminal prodomain. All mature polypeptides are structurally relatedand contain a conserved bioactive domain comprising six or sevencanonical cysteine residues which are responsible for thecharacteristical three-dimensional “cystine-knot” motif of theseproteins.

The various superfamily members can be further classified into distinctsubfamilies and -groups, based on the extent of the homology/identity oftheir cystine-knot motif. The overlapping families of bone morphogeneticproteins and growth/differentiation factors (GDFs) are known to play adiverse set of roles in the skeletal system and other tissues (see i.e.Ducy and Karsenty 2000, Kidney Int. 57, 2207-2214 for a review).Especially human GDF-5 (the protein is also known as MP52, CDMP-1 orsometimes as BMP-14), GDF-6 (CDMP-2, BMP13) and GDF-7 (CDMP-3, BMP-12)have been grouped together by several authors due to their comparablebiological properties and the extraordinarily high degree of amino acidsequence identity (see i.e. Mikic 2004, Annals of Biomedical Engineering32, 466-476; Wolfman et al. 1997, J. Clin. Invest. 100, 321-330).

Besides the prominent functions of the GDF-5/-6/-7 subgroup in the denovo formation of bone and cartilage (Cheng et al. 2003, J. Bone & JointSurg. Am. 85-A, 1544-1552; Settle et al. 2003, Developm. Biol. 254,116-130), it has repeatedly been demonstrated that the members of thissubgroup are also important inducers and regulators of tendon andligament (Wolfman et al. 1997, J. Clin. Invest. 100, 321-330), nervetissues (Farkas et al. 1997, Neurosci Lett. 236, 120-122; Watakabe etal. 2001, J. Neurochem. 76, 1455-1464), periodontal ligament and teeth(Sena et al 2003, J. Dent. Res. 82, 166-171; Morotome et al. 1998,Biochem. Biophys. Res. Commun. 244, 85-90), and other tissues.

The gene and protein structures of various naturally occurring BMPs/GDFsincluding GDF-5, GDF-6 and GDF-7 have previously been elucidated.Several loss-of-function mutants of GDF-5 could be identified which i.e.lead to shortening of fingers and toes (brachydactyl)-type C) and otherskeletal abnormalities such as brachypodism in animals (Storm et al.1994, Nature 368, 639-643) and acromesomelic displasias in man (Thomaset al. 1996, Nature Gen. 12, 315-317). Regarding these mutants it hasbeen found that specific amino acid substitutions at positions 173, 204,400, 438, 441 and 498 of human GDF-5 either reduce or completely abolishthe protein function (Schwabe et al. 2004, Amer. J. Med. Genet. 124A,356-363). In contrast, only very few GDF-mutants with enhancedbiological activity are known to date. A rare example is disclosed inWO01/11041 and relates to active monomeric GDF-5 which lacks thecysteine residue normally responsible for dimerization.

The search for the molecules responsible for bone-, cartilage-, andother tissue-inductive activity has led to the discovery of a set ofmolecules called growth/differentiation factors. Due to their uniquetissue inductive activities these proteins have been successfullyapplied in therapeutic research and regenerative surgery in which theypromote and assist the natural healing process of damaged tissues,either alone or in combination with specific carrier and/or matrixmaterials. Nevertheless there is a great need to develop improved andmore efficient forms of these proteins for such purposes.

This object is solved according to the invention by providing novelrecombinant proteins derived from GDF-5-related proteins which exhibitimproved biological activity as described herein and in the attachedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, shows additional features of the human GDF-5 precursor proteinaccording to SEQ ID NO: 1.

FIG. 2 shows a comparison of the 102 aa cystine-knot-domains of humanGDF-5 (SEQ ID NO: 1), human GDF-6 (sequence 2 from U.S. Pat. No.5,658,882).

FIG. 3 shows a comparison of the 102 aa cystine-knot-domains ofvertebrate GDF-5, -6 and -7 sequences from the genus Homo, furtherCercopithecus, Macaca, Bos, Mus, Gallus Danio and Xenopus, which areavailable in the “Entrez” NCBI protein database(www.ncbi.nlm.nih.gov/Entrez/) under the accession numbers is shown inthe figure.

FIG. 4 lists the sequence identities of cystine-knot-domains of knownBMPs and GDFs to the cystine-knot-domain of human GDF-5.

FIG. 5 shows the results of an alkaline phosphatase assay (ALP) withrecombinant human GDF-5 (rh-GDF-5) and hGDF-5 mL-mutant M453V/M456V (asdescribed in example 2).

FIG. 6 shows histological cross-sections (AlizarinRed-S staining ofnewly formed calcium) and μCT scans of growth factor-treated scaffolds 4weeks after implantation in SCID mice according to example 3.

FIG. 7 shows an estimation of new bone formation on the scaffoldsaccording to example 3 is made. In each group, three animals wereexamined (n=3) and a cumulative value determined.

DETAILED DESCRIPTION OF THE INVENTION

In order to avoid misunderstandings and ambiguities, some frequentlyused terms herein are defined and exemplified as follows:

The term “cystine-knot-domain” as used herein means the well known andconserved cysteine-rich amino acid region which is present in the matureparts of TGF-beta superfamily proteins such as human GDF-5 and whichforms a three-dimensional protein structure known as cystine-knot. Inthis domain, the respective location of the cysteine residues to eachother is important and is only allowed to vary slightly in order not tolose the biological activity. Consensus sequences for cystine-knotdomains are known in the state of the art. According to the definitiondefined herein the cystine-knot-domain of a protein starts with thefirst cysteine residue participating in the cystine-knot of therespective protein and ends with the residue which follows the lastcysteine participating in the cystine-knot of the respective protein.For example, the cystine-knot domain of the human GDF-5 precursorprotein (SEQ ID NO 1) comprises the amino acids 400-501 (see also FIG.1).

The term “GDF-5-related protein” as used herein means any naturallyoccurring or artificially created protein which comprises acystine-knot-domain with an amino acid identity of at least 60% to the102 aa cystine-knot domain of human GDF-5 (amino acids 400-501 of FIG.1/SEQ ID NO 1) and which carries a methionine residue at a positionequivalent to residue methionine 453 (M453) of human GDF-5, and amethionine or leucine residue at a position equivalent to methionine 456(M456) of human GDF-5. Included are proteins belonging to the group ofGDF-5, GDF-6 and GDF-7 proteins from vertebrate or mammalian species aswell as recombinant variants thereof as long as these proteins fulfilthe above mentioned requirements.

Non-limiting examples of GDF-5-related proteins according to thedefinition above are human GDF-5 (disclosed as MP52 in WO95/04819 and inHötten et al. 1994, Biochem. Biophys Res. Commun. 204, 646-652),recombinant human GDF-5/MP52 (WO96/33215), mouse GDF-5 (U.S. Pat. No.5,801,014), CDMP-1 (WO96/14335), HMW human MP52s (WO97/04095), rabbitGDF-5 (Sanyal et al. 2000, Mol. Biotechnol. 16, 203-210), humanGDF-6/BMP-13 (U.S. Pat. No. 5,658,882), bovine GDF-6 (NCBI accession noP55106), mouse GDF-6 (NCBI accession no NP_(—)038554), GDF-6/CDMP-2(WO96/14335), human GDF-7/BMP-12 (U.S. Pat. No. 5,658,882), mouse GDF-7(NCBI accession no AAP97721), GDF-7/CDMP-3 (WO96/143335), chicken GDF-5(NCBI accession no. NP_(—)989669), Xenopus laevis GDF-5 (NCBI accessionno. AAT99303), monomeric GDF-5, -6 and -7 (WO 01/11041 and WO99/61611),as shown in FIGS. 3 and 4.

The term “ML-mutant” as used herein means a recombinant protein derivedfrom a GDF-5-related protein in which, after alignment with human GDF-5as described in this application, the amino acid equivalent tomethionine 453 (M453) of human GDF-5 is not methionine, and/or the aminoacid equivalent to methionine 456 (M456) of human GDF-5 (SEQ ID NO 1) isnot methionine (M) or leucine (L).

The term “improved biological activity” as used herein relates to abiological activity of a ML-mutant amounting to at least 120% of theactivity of the respective non-mutated protein.

The term “biological activity” denotes the activity of a GDF-5-relatedprotein as measured by one or more of the following assays:

a) an in vitro alkaline phosphatase assay (ALP), e.g. as described inTakuwa et al. (1989), Am. J. Physiol. 257, E797-E803);b) measurement of increased survival of dopaminergic neurons asdescribed for example by Krieglstein et al. 1995 (J. Neuroscience Res.42, 724-732) or Sullivan et al. 1997 (Neuroscience Letters 233, 73-76);c) the outgrowth of nerve fibers from embryonic retina as measured e.g.as described i.e. in WO97/03188;d) the angiogenic potential of these proteins as verified for example inan in vivo corneal micropocket model as described in Yamashita et al.1997 (Exp. Cell Research 235, 218-226);e) effects of GDF-5-related proteins on the terminal differentiation ofmyoblasts as determined as described e.g. by Inada et al 1996 (BiochemBiophys Res Commun. 222, 317-322);f) in vivo tests measuring the inductive potential of such proteinsconcerning tendon and ligament e.g. as disclosed in Wolfman et al. 1997,J. Clin. Invest. 100, 321-330;g) measurement of the signal transduction cascade through the activationof Smads using a reportergene assay based on the Smad-binding-elementspreceding the firefly luciferase gene e.g. as previously described (Noheet al., 2002. J Biol. Chem. 277, 5330-5338.)

The term “variant” as used herein means any of the followingpolypeptides:

a) biologically active fragments of a proteinb) protein constructs which contain additional sequences in excess tothe original sequence of the proteinc) any combination of a) and b)

The GDF-5/-6/-7 group of TGF-beta superfamily proteins, comprising GDF-5as its best characterized member, is highly conserved amongvertebrate/mammalian species (Ducy and Karsenty 2000, Kidney Int. 57,2207-2214). It has now surprisingly been found by means of mutationalstudies and other experiments that amino acid residues which correspondto methionine 453 (M453) and methionine 456 (M456) of human GDF-5 can besubstituted with some specified amino acids without negative effects onthe protein function. Moreover, these substitutions even increase thebiological activity of the proteins significantly.

This embodiment of the invention is further illustrated by the FIGS. 1,2 and 3. FIG. 1 shows the human GDF-5 precursor protein (Hötten et al.1994, Biochem. Biophys Res. Commun. 204, 646-652) which consists of a381 aa prodomain (aa 1-381 including signal peptide (aa 1-27), boldletters) and a 120 aa mature part (aa 382-501). The mature part orespecially the cystine-knot-domain (aa 400-501, underlined) aresufficient for the biological function of the protein. Residues M453 andM456 (grey boxes) are located within this cystine-knot domain.Corresponding residues in the cystine-knot-domains of otherGDF-5-related proteins are shown in FIG. 2 and FIG. 3 (marked byarrows). Corresponding residues in proteins not shown in these figurescan be easily determined by a sequence alignment with human GDF-5.

It has been found in GDF-5-related proteins that when the methionineresidue at a position corresponding to methionine 453 (M453) of humanwild-type GDF-5 (SEQ. ID NO 1) is replaced with an amino acid chosenfrom alanine (A), valine (V) or isoleucine (I), the resultingrecombinant protein has increased biological activity.

In a preferred embodiment, the chosen amino acid is valine (V) for theposition M453.

It has also been found that when the methionine residue at a positioncorresponding to methionine 456 (M456) of human wild-type GDF-5 (SEQ. IDNO 1) is replaced with an amino acid chosen from alanine (A), valine (V)or isoleucine (I), either independently, or in combination with areplacement of M453, the resulting recombinant protein has increasedbiological activity.

In a preferred embodiment, the chosen amino acid is valine (V) for theposition M456.

These ML-mutants of GDF-5-related proteins in which the M453 and/or M456equivalents are substituted with the amino acids specified above exhibita biological activity greatly outperforming the activity of therespective nonmutated proteins.

As an example, FIG. 5 shows the enhanced ability of hGDF-5 mL-mutantM453V/M456V to induce alkaline phosphatase in vitro. The mutant proteinexhibits a biological activity of 585.5% (at 133 ng/ml), 356.3% (at 400ng/ml) and 236.3% (at 1200 ng/ml) of the activity of wildtype protein(rh-GDF-5) in this assay (average of multiple experiments). Thus, theaverage activity is 585.5+356.3+236.3:3=392.7% of the activity ofwildtype protein (rh-GDF-5). The minimal activity measured for themutant at a single protein concentration and in a single experiment was150% of the activity of the wild-type protein.

Thus, encompassed by the invention are ML-mutants which exhibit animproved biological activity amounting to at least 120% of the activityof the respective non-mutated protein. Especially preferred areGDF-5-related ML-mutants with improved biological activities of at least150%, preferably 160%, more preferably at least 170%, more preferably atleast 180%, and most preferably at least 200% of the biological activityof the respective non-m mutated protein.

The biological activities of GDF-5-related proteins and ML-mutantsthereof i.e. in the field of induction of bone, cartilage and connectivetissue such as i.e. periodontal ligament can be easily determined withthe help of established test systems. Most useful and preferred is acommon in vitro test known as alkaline phosphatase (ALP) assay (Takuwaet al. 1989, Am. J. Physiol. 257, E797-E803), which is also demonstratedin example 2/FIG. 5. GDF-5-related proteins have been demonstrated toincrease alkaline phosphatase activity i.e. in ROB-C26 osteoprogenitorcells (Yamaguchi et al. 1991, Calcif. Tissue Int. 49, 221-225) asdescribed in WO95/04819, in embryonic ATDC5 cells (Riken Gene Bank, ROB0565), in mouse stromal MCHT-1/26 cells, and in periodontal ligament(HPDL) cells as shown in Nakamura et al. 2003, J. Periodontal Res. 38,597-605.

The GDF-5-related proteins as defined herein comprise acystine-knot-domain with an amino acid identity of at least 60%,preferably at least 75%, more preferably at least 80%, more preferablyat least 90%, and most preferably at least 95%, to the 102 aacystine-knot domain of human GDF-5. A limiting value of 60% is wellsuitable to separate members of the GDF-5/-6/-7 group of proteins aswell as variants thereof from further proteins such as other GDFs andBMPs. A comparison of the 102 aa cystine-knot-domains of human GDF-5,human GDF-6 and human GDF-7 (FIG. 2) reveals the high grade of aminoacid identity between these proteins. Human GDF-6 shares 87 (85%) andhuman GDF-7 83 (81%) identical residues with the cystine-knot-domain ofhuman GDF-5. The respective domains of GDF-5/-6/-7 molecules from othervertebrate and mammalian species which have been identified so far alsoshow very high identity percentages of at least 75% (between 79% and99%), when compared with human GDF-5 (FIG. 4). In contrast, GDFs andBMPs not belonging to the GDF-5/-6/-7 subgroup display much loweridentity values below 60%.

The determination of corresponding amino acid positions in related aminoacid sequences as well as the calculation of percentages of identitybetween can be performed with the help of well known alignmentalgorithms and optionally computer programs using these algorithms. Theamino acid identities in this patent application have been calculated byaligning sequences with the freeware program ClustalX (Version 1.81)with default parameters and subsequent counting of identical residues byhand. Default settings for pairwise alignment (slow-accurate) are: gapopening parameter: 10.00; gap extension parameter 0.10; Protein weightmatrix: Gonnet 250. The ClustalX program is described in detail in:Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. andHiggins, D. G. (1997)

The ClustalX windows interface: flexible strategies for multiplesequence alignment aided by quality analysis tools.

Nucleic Acids Research 24:4876-4882.

ClustalX is a windows interface for the ClustalW multiple sequencealignment program and is i.e. available from various sources, i.e. byanonymous ftp from ftp-igbmc.u-strasbg.fr, ftp.embl-heidelberg.de,ftp.ebi.ac.uk or via download from the following webpage:http://www-igbmc.u-strasbg.fr/BioInfo/. The ClustalW program andalgorithm is also described in detail in:

Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994)

CLUSTALW: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, positions-specific gap penaltiesand weight matrix choice. Nucleic Acids Research 22:4673-4680.

The ML-mutants according to the invention which are derived fromGDF-5-related proteins are generally applicable in every indication inwhich GDF-5-related proteins such as GDF-5, GDF-6 and GDF-7 are alsouseful. It has been demonstrated that GDF-5-related proteins areimportant inducers and regulators/differentiators of i.e. bone andcartilage (Cheng et al. 2003, J. Bone & Joint Surg. Am. 85-A, 1544-1552;Settle et al. 2003, Developm. Biol. 254, 116-130), connective tissuesuch as tendon and ligament (Wolf man et al. 1997, J. Clin. Invest. 100,321-330), nerve tissue (Farkas et al. 1997, Neurosci Lett. 236, 120-122;Watakabe et al. 2001, J. Neurochem. 76, 1455-1464), stem cells (Shimaokaet al. 2003, J. Biomed. Materials Res. Part A 68A, 168-176; Bai et al.2004, Biochem. Biophys. Res. Commun. 325, 453-460) and/periodontalligament and teeth (Sena et al 2003, J. Dent. Res. 82, 166-171; Morotomeet al. 1998, Biochem. Biophys. Res. Commun. 244, 85-90).

In a preferred embodiment, the ML-mutant comprises a sequence whichmatches one of the following generic amino acid sequences

a)CX₁X₂KX₃LHVX₄FX₅X₆X₇GWDDWX₈IAPLX₉YEAX₁₀HCX₁₁GX₁₂CX₁₃FPX₁₄RSHLEPTNHAX₁₅IQTLZ₁NSMX₁₆PX₁₇X₁₈X₁₉PX₂₀₁X₂₁CCVPX₂₂X₂₃LX₂₄PISILX₂₅X₂₆DX₂₇X₂₈NNV VYX₂₉X₃₀YEX₃₁MVVEX₃₂CGCR orb)CX₁X₂KX₃LHVX₄FX₅X₆X₇GWDDWX₈IAPLX₉YEAX₁₀HCX₁₁GX₁₂CX₁₃FPX₁₄RSHLEPTNHAX₁₅IQTLMNSZ₂X₁₆PX₁₇X₁₈X₁₉PX₂₀₁X₂₁CCVPX₂₂X₂₃LX₂₄PISILX₂₅X₂₆DX₂₇X₂₈NNVVYX₂₉X₃₀YEX₃₁MVVEX₃₂CGCRorc)CX₁X₂KX₃LHVX₄FX₅X₆X₇GWDDWX₈IAPLX₉YEAX₁₀HCX₁₁GX₁₂CX₁₃FPX₁₄RSHLEPTNHAX₁₅IQTLZ₁NSZ₂X₁₆PX₁₇X₁₈X₁₉PX₂₀₁X₂₁CCVPX₂₂X₂₃LX₂₄PISILX₂₅X₂₆DX₂₇X₂₈NNVVYX₂₉X₃₀YEX₃₁MVVEX₃₂CGCR,and whereinevery X denotes any amino acid,Z₁ denotes alanine (A), isoleucine (I), or valine (V),Z₂ denotes alanine (A), isoleucine (I), or valine (V).

In a more preferred embodiment, the ML-mutant comprises a sequence whichmatches one of the above mention generic amino acid sequences andwherein

-   X₁ denotes asparagine (N) or serine (S)-   X₂ denotes arginine (R) or lysine (K)-   X₃ denotes alanine (A), glutamine (Q), proline (P) or serine (S)-   X₄ denotes asparagine (N) or aspartic acid (D)-   X₅ denotes arginine (R) or lysine (K)-   X₆ denotes aspartic acid (D) or glutamic acid (E)-   X₇ denotes leucine (L) or methionine (M)-   X₈ denotes isoleucine (I) or valine (V)-   X₉ denotes aspartic acid (D) or glutamic acid (E)-   X₁₀ denotes histidine (H), phenylalanine (F) or tyrosine (Y)-   X₁₁ denotes aspartic acid (D) or glutamic acid (E)-   X₁₂ denotes leucine (L), methionine (M) or valine (V)-   X₁₃ denotes aspartic acid (D) or glutamic acid (E)-   X₁₄ denotes isoleucine (I) or leucine (L)-   X₁₅ denotes isoleucine (I) or valine (V)-   X₁₆ denotes alanine (A), asparagine (N) or aspartic acid (D)-   X₁₇ denotes arginine (R), asparagine (N), aspartic acid (D),    glutamic acid (E), glycine (G) or serine (S)-   X₁₈ denotes alanine (A), asparagine (N), serine (S) or threonine (T)-   X₁₉ denotes alanine (A), methionine (M) or threonine (T)-   X₂₀ denotes alanine (A) or proline (P)-   X₂₁ denotes serine (S) or threonine (T)-   X₂₂ denotes alanine (A), serine (S) or threonine (T)-   X₂₃ denotes arginine (R) or lysine (K)-   X₂₄ denotes serine (S) or threonine (T)-   X₂₅ denotes phenylalanine (F) or tyrosine (Y)-   X₂₆ denotes isoleucine (I) or threonine (T)-   X₂₇ denotes alanine (A) or serine (S)-   X₂₈ denotes alanine (A) or glyine (G)-   X₂₉ denotes asparagine (N) or lysine (K)-   X₃₀ denotes glutamic acid (E) or glutamine (Q)-   X₃₁ denotes aspartic acid (D) or glutamic acid (E),-   X₃₂ denotes alanine (A), glutamine (Q), serine (S) or threonine (T)-   Z₁ denotes alanine (A), isoleucine (I), or valine (V),-   Z₂ denotes alanine (A), isoleucine (I), or valine (V).

These generic sequences have been compiled from a comparison of thecystine-knot domains of vertebrate GDF-5, GDF-6 and GDF-7 sequencesaccording to FIG. 3. Positions which are not conserved in the alignedproteins are denoted with an X in the generic sequences. Positions whichare mutated according to the present invention are denoted with a Z.

In another preferred embodiment, the ML-mutant protein according to theinvention is an ML-mutant of a vertebrate or recombinant GDF-5 proteinor a variant thereof. Most preferred are ML-mutants of a mammalian GDF-5protein or variants thereof. Examples for vertebrate and mammalian GDF-5proteins are: human GDF-5 (disclosed as MP52 in WO95/04819 and as humanGDF-5 in Hötten et al. 1994, Biochem. Biophys Res. Commun. 204,646-652), recombinant human GDF-5/MP52 (WO96/33215), HMW human MP52s(WO97/04095), CDMP-1 (WO96/14335), mouse (Mus musculus) GDF-5 (U.S. Pat.No. 5,801,014), rabbit (Oryctolagus cuniculus) GDF-5 (Sanyal et al.2000, Mol Biotechnol. 16, 203-210), chicken (Gallus gallus) GDF-5 (NCBIaccession no. NP_(—)989669), African clawed frog (Xenopus laevis) GDF-5(NCBI accession no. AAT99303).

Another preferred embodiment of the invention includes ML-mutantproteins of monomeric GDF-5-related proteins. In these monomericproteins, the cysteine which is responsible for dimer formation iseither substituted by another amino acid or deleted. Such proteins aree.g. described in WO 01/11041 and WO 99/61611, which are herewithincorporated by reference. An especially preferred monomeric protein isrecombinant monomeric GDF-5 as disclosed therein.

Enclosed in these embodiments are also ML-mutants of allelic versions ofthe aforementioned genes/proteins as well as ML-mutants of thevertebrate, mammalian and recombinant proteins or variants thereofhaving additional mutations such as substitutions, additions anddeletions, as long as these additional mutations have no essentialeffect on protein activity.

In general, the ML-mutant of the vertebrate or mammalian or recombinantGDF-5 protein or variant thereof is expected to show all alreadydescribed activities of GDF-5 and can be applied wherever the abovementioned recombinant and wild-type GDF-5 forms are been successfullyused. For example, GDF-5 is considered to be a very effective promoterof bone and cartilage formation as well as connective tissue formation(see for example WO 95/04819, Hötten et al. 1996, Growth Factors 13,65-74; Storm et al. 1994, Nature 368, 639-643; Chang et al. 1994, J.Biol. Chem. 269, 28227-28234) and formation of connective tissueattachment (EP 0 831 884. In this context, GDF-5 is useful forapplications concerning the joints between skeletal elements (see forexample Storm & Kingsley 1996, Development 122, 3969-3979). One examplefor connective tissue is tendon and ligament (Wolfman et al. 1997, J.Clin. Invest. 100, 321-330; Aspenberg & Forslund 1999, Acta Orthop Scand70, 51-54; WO 95/16035). The protein is helpful for meniscus andspinal/intervertebral disk repair (Walsh et al. 2004, Spine 29, 156-63)and spinal fusion applications (Spiro et al. 2000, Biochem Soc Trans.28, 362-368). GDF-5 can be beneficially applied in tooth (dental andperiodontal) applications (see for example WO 95/04819; WO 93/16099;Morotome et al. 1998, Biochem Biophys Res Comm 244, 85-90) such as theregeneration of dentin or periodontal ligament.

GDF-5 is also useful in wound repair of any kind. It is also beneficialfor promoting tissue growth in the neuronal system and survival of e.g.dopaminergic neurons. In this context, GDF-5 can be used for treatingneurodegenerative disorders like e.g. Parkinson's disease and possiblyalso Alzheimer's disease or Huntington chorea tissues (see for exampleWO 97/03188; Krieglstein et al., (1995) J. Neurosci Res. 42, 724-732;Sullivan et al., (1997) Neurosci Lett 233, 73-76; Sullivan et al.(1998), Eur. J. Neurosci 10, 3681-3688). GDF-5 allows to maintainnervous function or to retain nervous function in already damagedtissues. GDF-5 is therefore considered to be a generally applicableneurotrophic factor.

It is also useful for diseases of the eye, in particular retina, corneaand optic nerve (see for example WO 97/03188; You et al. (1999), InvestOpthalmol V is Sci 40, 296-311), for hair growth and the treatment anddiagnosis of skin related disorders (WO 02/076494; Battaglia et al.2002, Trans. Orthop. Res. Soc. 27, 584), and for induction ofangiogenesis (Yamashita et al. 1997, Exp. Cell Res. 235, 218-26).

On the one hand, there is the prevention or therapy of diseasesassociated with bone and/or cartilage damage or affecting bone and/orcartilage disease, or generally situations, in which cartilage and/orbone formation is desirable or for spinal fusion, and on the other hand,there is prevention or therapy of damaged or diseased tissue associatedwith connective tissue including tendon and/or ligament, periodontal ordental tissue including dental implants, neural tissue including CNStissue and neuropathological situations, tissue of the sensory system,liver, pancreas, cardiac, blood vessel, renal, uterine and thyroidtissue, skin, mucous membranes, endothelium, epithelium, for promotionor induction of nerve growth, tissue regeneration, angiogenesis, woundhealing including ulcers, burns, injuries or skin grafts, induction ofproliferation of progenitor cells or bone marrow cells, for maintenanceof a state of proliferation or differentiation for treatment orpreservation of tissue or cells for organ or tissue transplantation, forintegrity of gastrointestinal lining, for treatment of disturbances infertility, contraception or pregnancy.

Diseases concerning sensory organs like the eye are also to be includedin the preferred indication of the pharmaceutical composition accordingto the invention. As neuronal diseases again Parkinson's and Alzheimer'sdiseases can be mentioned as examples.

Example 3 and FIG. 6 describe the results of an alkaline phosphataseassay with recombinant human GDF-5 (WO96/33215) and the ML-mutantM453V/M456V of recombinant human GDF-5 (rhGDF-5). Recombinant humanGDF-5 was used as a standard/control with 100% biological activity. Themutant protein exhibits a biological activity of 585.5% (at 133 ng/ml),356.3% (at 400 ng/ml) and 236.3% (at 1200 ng/ml) of the activity ofwildtype protein (rh-GDF-5) in this assay (average of multipleexperiments). Thus, the average activity is 585.5+356.3+236.3:3=392.7%of the activity of wildtype protein (rh-GDF-5). The minimal activitymeasured for the mutant at a single protein concentration and in asingle experiment was 150% of the activity of the wild-type protein.

The ML-mutants according to the invention can be easily produced invarious prokaryotic and eukaryotic expression systems, in particular byexpression in prokaryotes and subsequent renaturation/refoldingaccording to known methods (see i.e. WO96/33215).

A further subject matter of the present invention is a nucleic acidencoding an ML-mutant according to the invention. The nucleic acid has asequence such that a substitution of one or both residues equivalent toM453 and M456 of human GDF-5 with one of the amino acids specified inthis application is achieved. The base triplets coding for these aminoacids and the degeneracy of the genetic code are generally known. Thenucleic acid can be a DNA sequence and/or a RNA sequence, as long as theprotein according to the invention can be obtained from this nucleicacid upon expression in a suitable system.

Expression vectors are a further subject matter of the presentinvention, wherein the nucleic acid is inserted in a suitable vectorsystem, the vector system being selected according to the desiredexpression of the protein. The vector system can be a eukaryotic vectorsystem, but preferred is a prokaryotic vector system, with which theproteins can be produced in a particularly easy and pure manner. Asuitable expression vector is i.e. shown in WO96/33215. The expressionvector can also be a viral vector which can be used i.e. in gene therapyapproaches.

Host cells are also a subject matter of the present invention. The hostcells are characterized in that they contain a nucleic acid or anexpression vector according to the invention and that they are able touse the information present in the nucleic acids and in the expressionvector, respectively, for the expression of ML-mutants according to theinvention. Suitable host cells are preferably prokaryotic cells, inparticular most E. coli strains. Particularly useful host strains aredescendents of E. coli W3110 as shown e.g. in WO96/33215. In a preferredembodiment, host cells, preferably of human origin, may also be usefulfor transplantation to patients in need thereof.

Another subject matter of the present invention are antibodies againstML-mutants. These antibodies according to the present invention arespecific for the claimed recombinant ML-mutant. Preferably, they arespecific for the cystine knot regions of GDF-5-related proteinscontaining one or more of the amino acid replacements described herein.Preferably, the antibodies are specific for a region of a recombinantprotein derived from a GDF-related protein according to the inventionspanning amino acid 400-495, preferably 420-460, more preferably440-460, more preferably amino acids 453-456. These antibodies accordingto the present invention can be generated by using those fragments ofthe protein of the invention as described above as immunogens togenerate antibodies by known methods. The antibodies can be monoclonalor polyclonal and they can be of any isotype. Also comprised areantibody fragments such as Fab-fragments or Fab₂-fragments. Theantibodies can also be humanized antibodies or chimeric antibodies etc.

Further subject matters of the present application are pharmaceuticaland/or diagnostic compositions comprising at least one ML-mutant of aGDF-5-related protein or a nucleic acid or a vector or host cellaccording to the invention. Suitable are generally all pharmaceuticalcomposition which have already been published in context withGDF-5-related proteins. An expression vector or a host cell can beconsidered to be advantageous as active substances in a pharmaceuticaland/or diagnostic composition. Also combinations of a protein accordingto the invention with other proteins can be used in preferredpharmaceutical compositions. Especially preferred for neuronalapplications are combinations with other TGF-beta superfamily proteinssuch as i.e. GDNF (see WO 97/03188). For applications concerningcartilage and/or bone the combination with BMPs in general or with acartilage maintenance-inducing protein such as BMP-9 (see e.g. WO96/39170) is useful. Combinations with other proteins such as i.e. NGF,BDNF, EGF, PDGF, NT-3, -4, -5, chordin and/or hedgehog proteins are alsopossible (see i.e. WO97/03188). Of course this invention also comprisespharmaceutical compositions containing further substances like e.g.pharmacologically acceptable auxiliary and carrier substances. Theformulation may include antioxidants, preservatives, colouring,flavouring and emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, excipients and/orpharmaceutical adjuvants. For example, a suitable carrier or vehicle maybe water for injection, physiological saline solution, or a salinesolution mixed with a suitable carrier protein such as serum albumin. Apreferred antioxidant for the preparation of the composition of thepresent invention is ascorbic acid.

Cosmetic compositions known in the art, preferably hypoallergic and pHcontrolled are especially preferred, and include toilet waters, packs,lotions, skin milks or milky lotions. Said preparations contain, besidesthe active compound, components usually employed in such preparations.Examples of such components are oils, fats, waxes, surfactants,humectants, thickening agents, antioxidants, viscosity stabilizers,chelating agents, buffers, preservatives, perfumes, dyestuffs, loweralkanols, and the like. If desired, further ingredients may beincorporated in the compositions, e.g. antiinflammatory agents,antibacterials, antifungals, disinfectants, vitamins, sunscreens,antibiotics, or other anti-acne agents.

The solvent or diluent of the pharmaceutical composition may be eitheraqueous or non-aqueous and may contain other pharmaceutically acceptableexcipients which are capable of modifying and/or maintaining a pH,osmolarity, viscosity, clarity, scale, sterility, stability, rate ofdissolution or odour of the formulation. Similarily other components maybe included in the pharmaceutical composition according to the presentinvention in order to modify and/or maintain the rate of release of thepharmaceutically effective substance. Such modifying components aresubstances usually employed in the art in order to formulate dosages forparenteral administration in either unit or multi-dose form. The finallyformulated pharmaceutical and/or diagnostic composition preparedaccording to the present invention may be stored in sterile vials inform of a solution, suspension, gel, emulsion, solid or dehydrated orlyophilized powder. These formulations may be stored either in aready-to-use form or in a form, e.g. in case of a lyophilized powder,which requires reconstitution prior to administration. The above andfurther suitable pharmaceutical formulations are known in the art andare described in, for example, Gus Remington's Pharmaceutical Sciences(18th Ed., Mack Publishing Co., Eastern, Pa., 1990, 1435-1712). Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the pharmaceuticallyeffective component. Other effective administration forms compriseparenteral slow-release, i.e. retarded, formulations, inhalent mists, ororally active formulations. For example, a slow-release formulation maycomprise proteins bound to or incorporated into particulate preparationsof polymeric compounds (such as polylactic acid, polyglycolic acid,etc.) or liposomes. The pharmaceutical composition according to thepresent invention may also be formulated for parenteral administration,e.g., by infusion or injection, and may also include slow-release orsustained circulation formulations. Such parenterally administeredtherapeutic compositions are typically in the form of pyrogen-free,parenterally acceptable aqueous solutions comprising thepharmaceutically effective component(s) in a pharmaceutically acceptablecarrier and/or diluent.

The pharmaceutical composition may comprise a matrix material, i.e. incases where regeneration of bone or cartilage is intended. It isadvantageous to the protein, the nucleic acid, the expression vector orthe host cell when they are applied in and/or on a biocompatible matrixmaterial. Matrix material as used herein means a carrier or matrixacting as a scaffold for cell recruitment, attachment, proliferation anddifferentiation and/or as a potential delivery and storage device forML-mutants of GDF-5-related proteins. In contrast to the solid matrices,carriers consist of amorphous materials having no defined surfaces andlacking a specific shape, i.e. alkylcelluloses, pluronics, gelatins,polyethylene glycols, dextrins, vegetable oils, sugars and other liquidand viscous substances.

Uses of GDF-5-related proteins or similar morphogens suchs as BMPs incombination with matrix materials are extensively published anddescribed, such as for example in WO98/21972. These matrix materials areequally suitable for ML-mutants according to the invention. The matrixmaterial can be transplanted into the patient, e.g. surgically, whereinthe protein or the DNA encoding the protein can be slowly released fromthe matrix material and then be effective over a long period of time.All types of matrix materials are useful in accordance with the presentinvention, as long as they are biocompatible and selected for theintended area or indication of use. The matrix material can be a naturalmaterial, a modified natural material as well as a synthetic material.All already known matrices for morphogenetic proteins are encompassed.Examples of natural materials are e.g. autologous, heterologous orxenologous bone materials, collagen, e.g. collagen type I and III, ormetals like titanium. Also other components of the extracellular matrixcan be used. The extracellular matrix comprises for example the variouscollagens, as for example types I, II, V, IX, X, XI and XIII, furtherproteoglycanes and glycosaminoglycanes, as for examplechondroitinsulfate, biglycane, decorine and/or hyaluronic acid, ornoncollagenous proteins as for example osteopontin, laminin,fibronectin, vitronectin, thrombospondin, cartilage matrix protein anddentin phosphoprotein. All mentioned natural materials may also be usedin artificially modified forms. Examples of modified natural materialsare demineralized bone, thermoashed bone mineral, sintered bone orchemically crosslinked hyaluronic acid (hydrogel), or metal alloys.Examples of synthetic materials are polymers like polyglycolic acid,polylactide and polylactide derivatives such as e.g. polylactic acid,poly(lactide-co-glycolide), polylactid acid-polyethylene glycol orglycolide L-lactide copolymers, further polyphosphates, polyethyleneglycol, polyoxyethylene polyoxypropylene copolymers or materialscontaining calcium phosphates such as beta-tricalcium phosphate(Ca₃(PO₄)₂), alpha-tricalcium phosphate and hydroxyl apatite. Furtherexamples of other useful matrix materials belonging to one of the abovementioned groups are Ca(OH)₂, coral, natural bone mineral, chitin,non-demineralized bone particles, ceramic bone particles, ceramicdentin, irradiated cancellous bone chips, plaster of Paris, bioactiveglass, apatite-wollastonite-containing glass ceramic. Also a combinationof the above mentioned carriers and/or matrices can form the matrixmaterial as for example the combination of hydroxy apatite and collagen(e.g. Healos, previously available from Orquest, Inc., CA, USA, [nowDePuy Acromed, MA, USA]), a combination of polyglycolic acid andpolylactic acid or polylactid derivatives, or coral-collagen composites.For a non limiting list of useful carriers and matrices see further i.e.Kirker-Head 2000, Advanced Drug Delivery 43, 65-92.

The following non-limiting examples together with the figures andsequence protocols are intended to further illustrate the invention.

SEQ ID NOS 1 and 2 shows the DNA and protein sequence of the human GDF-5precursor. In the preferred human GDF-5 protein mutants with improvedbiological activity, the methionine residue at pos 453 and/or themethionine residue at pos 456 are substituted with other amino acids.

As shown in FIG. 1, additional features of the human GDF-5 precursorprotein according to SEQ ID NO 1 are designated as:

-   aa 001-381 pre-prodomain (bold letters)-   aa 382-501 mature protein part-   aa 400-501 cystine-knot-domain (underlined)-   aa 453 residue methionine 453 (grey box)-   aa 456 residue methionine 456 (grey box)

A comparison of the 102 aa cystine-knot-domains of human GDF-5 (SEQ IDNO 1), human GDF-6 (sequence 2 from U.S. Pat. No. 5,658,882) and humanGDF-7 (sequence 26 from U.S. Pat. No. 5,658,882) is made in FIG. 2.Amino acid residues which are identical in all three molecules arehighlighted in black. Residues M453 and M456 of human GDF-5 andequivalent residues of human GDF-6 and GDF-7 are marked by arrows.

A comparison of the 102 aa cystine-knot-domains of vertebrate GDF-5, -6and -7 sequences from the genus Homo, further Cercopithecus, Macaca,Bos, Mus, Gallus Danio and Xenopus, which are available in the “Entrez”NCBI protein database (www.ncbi.nlm.nih.gov/Entrez/) under the accessionnumbers is shown in FIG. 3. Residues M453 and M456 of human GDF-5 andequivalent residues of the other proteins are marked by arrows.

The sequence identities of cystine-knot-domains of known BMPs and GDFsto the cystine-knot-domain of human GDF-5 are listed in FIG. 4.

The results of an alkaline phosphatase assay (ALP) with recombinanthuman GDF-5 (rh-GDF-5) and hGDF-5 mL-mutant M453V/M456V (as described inexample 2) are illustrated in FIG. 5.

FIG. 6 shows histological cross-sections (AlizarinRed-S staining ofnewly formed calcium) and μCT scans of growth factor-treated scaffolds 4weeks after implantation in SCID mice according to example 3.

An estimation of new bone formation on the scaffolds according toexample 3 is made in FIG. 7. In each group, three animals were examined(n=3) and a cumulative value determined. The following scaling was used:

no bone 0 1-10% bone 1 10-50%  bone 2 50-100% bone 3

Example 1 Creation, Expression and Purification of ML-Mutants

DNAs coding for the mature parts of human GDF-5, human GDF-6 and humanGDF-7 proteins have been isolated from human ROB-C26 osteoprogenitorcells (Yamaguchi et al. 1991, Calcif. Tissue Int. 49, 221-225) viaRT-PCR technique and subsequently ligated into prokaryotic plasmidvectors. In order to identify functionally important amino acid residuesin the mature parts of GDF-5, -6 and -7, various single mutations havebeen introduced into these sequences via site directed mutagenesis. Allindividual mutations were created by using the QuickChange™site-directed mutagenesis kit with the PfuTurbo™ DNA polymerase and theDPN I endonuclease from Stratagene according to the instruction manualof the manufacturer.

Using the bacterial strain W3110BP transformed with the plasmids andinduced with IPTG, the proteins were expressed in inclusion bodies.These inclusion bodies were isolated using a homogenization buffer (25mM Tris HCl pH 7.3, 10 mM EDTA NaOH pH 8, 8 M Urea) and wash buffer (1 MUrea, 20 mM Tris HCl, pH 8.3, 10 mM EDTA NaOH pH 8.0) according tostandard procedures. Further purification was carried out on a reversedphase column Aquapore Octyl (Applied Biosys, (CV=7.8 ml) 100×10, 20μ, No186470) with a gradient from 100% of Eluent A (0.1% TFA, HPLC H₂O) to100% Eluent B (0.1% TFA, 90% CH₃N, HPLC H₂O) in 104 minutes (flow rate:3 ml/min). After a western blot control, the fractions containing themutant protein were pooled and lyophilized.

The mutant proteins were dissolved in dissolving buffer (6 M GuanidinHCl, 50 m M Tris, 150 mM NaCl, 3 mM DTT, pH=8.0), the proteinconcentration was exactly adjusted to 2.6 mg/ml and the pH was adjustedbetween 8 and 9. After 2 h incubation at room temperature, refoldingbuffer (1 M NaCl, 50 mM Tris, 5 mM EDTA, 1 mM GSSG, 2 mM GSH, 33 mMChaps, pH=9.5) was added under gentle agitation to reach a finalconcentration of 0.16 mg/ml.

The solution was then incubated for 48 h at 22° C. and the refolding wasstopped by changing the pH to 3-4 by adding 18% HCl. Aftercentrifugation, the non-refolded monomer was separated from the dimerform by carrying out a second RP-HPLC under the same conditions. Thefractions containing the dimerized protein were pooled, lyophilized andstored at −70° C.

Example 2 Measurement of the Biological Activity of ML-Mutants In Vitroby ALP Assay

1×10⁴ cells of osteo-/chondroprogenitor cell line ATDC-5 were incubatedovernight in 96-well plates in cell culture medium (alpha-MEM,Penicilline/Streptomycine, 2 mM L-glutamine, 10% FCS) at 37° C., 5% CO₂,H₂O-saturated. The next day, cells were stimulated with theGDF-5-related proteins and mutants thereof for 72 hrs with indicatedligand concentrations. The cells were subsequently washed with PBS(phosphate buffered saline). Cell lysis was performed in 100 μl alkalinelysis buffer 1 (0.1M glycine, pH 9.6, 1% NP-40, 1 mM MgCl₂, 1 mM ZnCl₂)for 1 h at room temperature. Then 100 μml alkaline lysisbuffer 2 wasadded (0.1M glycine, pH 9.6, 1 mM MgCl₂, 1 mM ZnCl₂+2 mg/ml PNPP). Theplates were incubated at 37° C., 5% CO₂, H₂O-saturated. The ALP-reactionwas stopped afterwards with 100 μl of 30 g/l NaOH and finally theoptical density was measured with an automatic microplate reader at 405nm under consideration of blank value subtraction.

As an example, results (average values of 2 independent experiments)regarding hGDF-5 mutant M453V/M456V are shown in FIG. 5. The mutantprotein exhibits a biological activity of 585.5% (at 133 ng/ml), 356.3%(at 400 ng/ml) and 236.3% (at 1200 ng/ml) of the activity of wildtypeprotein (rh-GDF-5) in this assay (average of multiple experiments). Theminimal activity measured for the mutant at a single proteinconcentration and in a single experiment was 150% of the activity of thewild-type protein.

Example 3 Ectopic Bone Formation In Vivo: a SCID-Mouse Model forrhGDF-5, rhGDF-5 M453V/M456V and BMP-2

The improved bone inducing capabilities of ML-mutants of GDF-5 relatedproteins were also verified in vivo. Beta-tricalcium phosphate ceramics(chronOS®, Synthes/RMS Foundation) were used as biodegradablebiomaterials with a size of 3×3×3.5 mm. Growth factors were dissolved insodium acetate pH 4, as followed: rhGDF-5 in a concentration of 10 μg in7 μl; rhGDF-5 M453V/M456V in a concentration of 10 μg in 9 μl and BMP-2(Induct OS Wyeth® Lot-no., 20603) was first dissolved in sterile waterin a concentration of 3 mg/ml and then dissolved in sodium acetate in aconcentration of 10 μg in 7 μl. Controls were saturated with 7 μl sodiumacetate. After coating the scaffolds were then dried for 10 minutes andstored at −20° Celsius. Before implantation, the scaffolds were loadedwith 10 μl fibronectin. In this study, severe combined immunodeficientmice (SCID), (30+/−2 g) were used. Under general i.p. anaesthesia, onesubcutaneous pocket were bluntly created through a one centimeterincision at the back. One loaded scaffold was inserted into the pocket.The wound was closed with single interrupted sutures. Animals weresacrificed after four weeks and scaffolds were harvested. Histology andμCT scans (μCT 80 SCANCO MEDICAL) were performed. For histology,scaffolds were embedded in paraffine wax and sections of 5 μm thicknesswere stained with AlizarinRed-S (0.5%) and Fast Green (0.04%) todemonstrate the new built calcium within the scaffolds.

Results are displayed in FIGS. 6 and 7. Controls had no sign of newectopic bone formation. Scaffolds loaded with rhGDF-5 did show a mediumamount of newly built bone with some bone formation on the scaffold.RhGDF-5 M453V/M456V had the highest value of newly bone formation.Scaffolds loaded with rhGDF-5 453V/M456V showed prominent bone on thescaffold. The scaffolds treated with BMP-2 showed prominent bone on thescaffolds but without being as homogeneous as the scaffolds loaded withrhGDF-5 453V/M456V. In summary, results confirmed that rhGDF-5453V/M456V leads to strongly enhanced bone formation in this SCID mousemodel.

1. A method for preventing or treating diseases which are associatedwith tissue degeneration and/or tissue destruction, in a patient in needthereof, comprising administering to the patient an effective amount ofa recombinant protein derived from a GDF-5-related protein, or a variantof the GDF-5-related protein, wherein a) the amino acid at the positioncorresponding to methionine 453 (M453) of human wild-type GDF-5 (SEQ IDNO 1) is alanine, valine or isoleucine, and/or b) the amino acid at theposition corresponding to methionine 456 (M456) of human wild-type GDF-5(SEQ ID NO 1) is alanine, valine or isoleucine, wherein the variant is abiologically active fragment, and wherein the GDF-5-related protein isany naturally occurring or artificially created protein which comprisesa cysteine-knot domain with an amino acid identity of at least 75% tothe 102 amino acid cysteine-knot domain of GDF-5, and further whereinthe recombinant protein has an improved biological activity of at least120% when compared to the mature GDF-5 protein.
 2. A method for thediagnosis, prevention and/or therapy of injuries or diseases associatedwith damaged bone, cartilage, connective tissue, connective tissueattachment, tendon, ligament, spinal/intervertebral disk, meniscus,dental tissue, dentin, periodontal ligament, blood vessels, skin, hairor neural tissue, in a patient in need thereof, comprising administeringto the patient an effective amount of a recombinant protein derived froma GDF-5-related protein, or a variant of the GDF-5-related protein,wherein a) the amino acid at the position corresponding to methionine453 (M453) of human wild-type GDF-5 (SEQ ID NO 1) is alanine, valine orisoleucine, and/or b) the amino acid at the position corresponding tomethionine 456 (M456) of human wild-type GDF-5 (SEQ ID NO 1) is alanine,valine or isoleucine, wherein the variant is a biologically activefragment, and wherein the GDF-5-related protein is any naturallyoccurring or artificially created protein which comprises acysteine-knot domain with an amino acid identity of at least 75% to the102 amino acid cysteine-knot domain of GDF-5, and further wherein therecombinant protein has an improved biological activity of at least 120%when compared to the mature GDF-5 protein.
 3. A method for promotingcartilage and/or bone growth and/or spinal fusion, for the diagnosis,prevention and/or therapy of damaged or diseased tissues selected fromthe group consisting of tissues of the sensory system, liver, pancreas,cardiac, blood vessel, renal, uterine and thyroid tissue, skin, mucousmembranes, endothelium, epithelium; for the induction of nerve growth,tissue regeneration, angiogenesis, wound healing including ulcers,burns, injuries and/or skin grafts; for the induction of proliferationof progenitor cells and/or bone marrow cells; for maintenance of a stateof proliferation or differentiation for treatment or preservation oftissue or cells for organ or tissue transplantation; for the treatmentof degenerative disorders concerning the joints to skeletal elementsand/or for meniscus and/or spinal/intervertebral disk repair; for themanufacture of a therapeutic and/or diagnostic composition for theprevention and/or treatment of neurodegenerative disorders, in a patientin need thereof, comprising administering to the patient an effectiveamount of a recombinant protein derived from a GDF-5-related protein, ora variant of the GDF-5-related protein, wherein a) the amino acid at theposition corresponding to methionine 453 (M453) of human wild-type GDF-5(SEQ ID NO 1) is alanine, valine or isoleucine, and/or b) the amino acidat the position corresponding to methionine 456 (M456) of humanwild-type GDF-5 (SEQ ID NO 1) is alanine, valine or isoleucine, whereinthe variant is a biologically active fragment, and wherein theGDF-5-related protein is any naturally occurring or artificially createdprotein which comprises a cysteine-knot domain with an amino acididentity of at least 75% to the 102 amino acid cysteine-knot domain ofGDF-5, and further wherein the recombinant protein has an improvedbiological activity of at least 120% when compared to the mature GDF-5protein.
 4. The method according to claim 3, wherein saidneurodegenerative disorder is selected from the group consisting ofParkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis(ALS), Multiple sclerosis and Huntington's disease.
 5. Method accordingto claim 4, wherein said disease associated with bone and cartilagedamage is osteoporosis.
 6. The method according to claim 3, wherein thedamaged or diseased tissue is selected from the group consisting ofconnective tissue including tendon and/or ligament, periodontal ordental tissue including dental implants, neural tissue including CNStissue and neuropathological situations, tissue of the sensory system,liver, pancreas, cardiac, blood vessel, renal, uterine and thyroidtissue, skin, mucous membranes, endothelium, epithelium.
 7. The methodaccording to claim 3, wherein the tissue is the gastrointestinal lining.8. The method according to claim 3, wherein the disease is associatedwith disturbances in fertility, contraception and/or pregnancy.
 9. Themethod according to claim 3, wherein the therapy is associated withpromoting hair growth.